Comparative Analysis of Volatile Defensive Secretions of Three Species of Pyrrhocoridae (Insecta: Heteroptera) by Gas Chromatography-Mass Spectrometric Method

Abstract

The true bugs (Hemiptera: Heteroptera) have evolved a system of well-developed scent glands that produce diverse and frequently strongly odorous compounds that act mainly as chemical protection against predators. A new method of non-lethal sampling with subsequent separation using gas chromatography with mass spectrometric detection was proposed for analysis of these volatile defensive secretions. Separation was performed on Rtx-200 column containing fluorinated polysiloxane stationary phase. Various mechanical irritation methods (ultrasonics, shaking, pressing bugs with plunger of syringe) were tested for secretion sampling with a special focus on non-lethal irritation. The preconcentration step was performed by sorption on solid phase microextraction (SPME) fibers with different polarity. For optimization of sampling procedure, Pyrrhocoris apterus was selected. The entire multi-parameter optimization procedure of secretion sampling was performed using response surface methodology. The irritation of bugs by pressing them with a plunger of syringe was shown to be the most suitable. The developed method was applied to analysis of secretions produced by adult males and females of Pyrrhocoris apterus, Pyrrhocoris tibialis and Scantius aegyptius (all Heteroptera: Pyrrhocoridae). The chemical composition of secretion, particularly that of alcohols, aldehydes and esters, is species-specific in all three pyrrhocorid species studied. The sexual dimorphism in occurrence of particular compounds is largely limited to alcohols and suggests their epigamic intraspecific function. The phenetic overall similarities in composition of secretion do not reflect either relationship of species or similarities in antipredatory color pattern. The similarities of secretions may be linked with antipredatory strategies. The proposed method requires only a few individuals which remain alive after the procedure. Thus secretions of a number of species including even the rare ones can be analyzed and broadly conceived comparative studies can be carried out.

Analysis of defensive secretion of a milkweed bug Lygaeus equestris by 1D GC-MS and GC×GC-MS: sex differences and host-plant effect

“…Because of the usage of the same sampling method, we could directly compare our results with those reported by Krajíček et al 11 for three true bug species from the family Pyrrhocoridae. Analytes belonging to the most abundant and thus probably representing the most important components of the defensive secretion of L. equestris were also identified in at least one of the three pyrrhocorid species analysed by Krajíček et al 11 . These analytes were (E)-2-octenal, decanal and (E)-2-octen-1-ol.…”

“…For the pilot sampling of the defensive secretion of L. equestris males raised on sunflower seeds and its subsequent GC analysis, we employed the methodology described by Krajíček et al 11 . As the sampling method by SPME fibre yielded peaks with very high response intensity, only one true bug individual was used for collection of the secretion, instead of three, as described by Krajíček et al 11 . The sample was separated on a medium polar Rtx-200 column, which is considered to be universal.…”

“…A non-lethal mechanical irritation method 11 was used for sampling the defensive secretion. The bugs were stressed by pressing them with the syringe plunger, which regularly resulted in release of the scent-gland secretion.…”

“…an attack of a vertebrate predator such as a bird or a lizard, and involves negligible loss of volatile compounds. The method was successfully applied for analysing defensive secretions of three true species of pyrrhocorid bugs by gas chromatography with mass spectrometric detection (GC-MS), combined with a mid-polar column (Rtx-200) 11 .…”

“…In this study, we analyzed the secretions of metathoracic scent glands in males and females of the milkweed bug L. equestris by 1D GC-MS and GC×GC-MS in combination with a commercial cryogenic modulator. A non-lethal method of mechanical irritation of the true bugs and solid phase microextraction (SPME) 11 were used for secretion sampling. Specifically, we compared the composition of secretion between bugs raised either on a toxic, cardenolide-containing host plant A. vernalis or on non-toxic sunflower as a control to test whether there are any intraspecific differences in composition of the secretion connected with feeding on different host plants and between sexes that might indicate a potential trade-off between the defense based on autogenous scent-gland secretion and the defense based on sequestered host-plant cardenolides.…”